1. Field of the Invention
The present invention relates generally to LC/MS analysis systems. More particularly the present invention relates to tracking entities from one injection to another during LC/MS experiments
2. Background of the Invention
A key problem in analytical chemistry is the estimation of the concentration of one or more molecular entities contained within a complex mixture. Liquid chromatography (LC) followed by mass spectrometry (MS) is a well-known technique (LC/MS) that can be used to separate large numbers of chemical entities in a sample to facilitate measuring concentration or quantity of each chemical entity. Measuring the exact mass of an entity allows the entity to be tracked between samples. Measuring the response or intensity of the tracked entity allows the concentration of an entity to be tracked from sample to sample.
In LC/MS, a sample is injected into the system for analysis. For each such injection, the LC/MS system measures the retention time, molecular weight, and intensity of ions. Multiple ions may arise from a single molecule. The concentration of the molecule can be determined by examination by one or more of the ions it produces.
As used herein, the term “entity” can mean a single ion from a molecule or the set of ions obtained from a single, common molecule. For example a small molecule of low molecular weight can produce a single ion. A large molecule, such as a peptide or a protein, can produce multiple ions. Well-known techniques can be used to combine multiple ions from a molecule to obtain a single effective, mass, retention time, and intensity. It is assumed that each entity has a mass, retention time, and intensity, and that an effective mass, retention time, and intensity can be assigned to each entity.
Using these measurements of mass, retention time, and intensity, properties of the entities can be determined. For example, comparison of intensities of corresponding entities between injections is the basis of determining whether the concentration of an entity changes between control and unknown samples. Changes in a protein's concentration between samples are indicative of changes in expression level of the protein between samples.
A set of samples can be processed using sequential injections. The same sample can be injected multiple times to provide a set of replicate injections. For example, each of two distinct samples (a standard and an unknown) can be injected three times, thereby producing a total of six injections. Using this data, reproducibility of the concentration measurements can be inferred for each entity, as well as the change in concentration of each entity between the control sample and the unknown sample. Each sample may contain an amount of an internal standard to provide a relative calibration between samples.
For a technique to determine the concentration of any entity, it must first adequately resolve that entity from all others. The LC/MS technique allows for separation of entities (or the ions associated with an entity) in both mass and retention time. Entities that co-elute in retention time, which would otherwise be indistinguishable, can be resolved in mass, thus allowing for their detection and for an accurate estimate of their intensity.
However, for associating or tracking an entity from one injection to another, resolution by accurate mass alone may not be sufficient. For example, consider the properties of mass and retention time of a molecule. The molecular weight is an intrinsic property of a molecule. A mass spectrometer measures the ratio of the molecular weight to charge, m/z. The symbol μ is often used to indicate the mass-to-charge ratio, m/z. Values for μ can be compared directly between injections. Any variation in measured values of μ between injections for the same entity must be due only to instrumental noise sources.
Ionization techniques, such as electrospray ionization may allow determination of charge, Z, for samples such as peptides or proteins. The determined charge state allows inference of the molecular weight, m, of an entity. Consequently, molecular weight, m, provides a basis for tracking entities. For these purposes, the empirically observed mass-to-charge ratio value, μ, or the inferred value of molecular weight, m, can be used, interchangeably. As used herein the term mass means the observed mass-to-charge ratio value, μ or the inferred molecular weight m.
With sufficiently high mass accuracy, each entity is potentially uniquely distinguishable based upon its value for mass. Thus, for a sample containing few entities, assuming sufficient chromatographic resolution to separate entities, a high accuracy mass spectrometer, such as a time-of-flight (TOF) analyzer with resolution of m/Δm≈20,000, allows tracking of each entity from one injection to another based upon accurate measurements of mass alone. In such cases, mass is not necessarily being used to identify an entity in terms of its chemical composition or structure. Rather, mass is being used as an empirical and possibly unique identifier of the entity to track the entity between injections.
However, mass alone may not be sufficient to track an entity from one injection to another. If mass accuracy is low and the sample complex, then it is possible that the mass of an entity as seen in one injection may match the empirically observed mass of an unrelated entity in another injection. For example, there may be two entities where μ is 1024.200 amu and 1024.300 amu respectively. While such entities are distinguishable with MS accuracy less than 0.100 amu, they are not distinguishable using MS having accuracy greater than 0.100 amu.
The chromatographic retention time of an entity can be an additional, potentially independent identifier of that entity. An entity's retention time is not an intrinsic property. Rather, an entity's retention time depends on the interactions of the entity (or, rather the molecule that gives rise to the entity) with the liquid and solid phases in the chromatographic separation, among other effect. But, even though the retention time is not intrinsic, its value can be made highly reproducible for a given separation method. Ideally, if the retention time were exactly reproducible and to high accuracy, then the combination of agreement in both mass and retention time could well be sufficient to allow each entity to be uniquely tracked from one injection to another. That is, it would be highly unlikely that two different entities share the exact same mass and retention time. However, retention time is not exactly reproducible between injections. Rather, the retention time of an entity can wander from injection to injection.
Despite such retention time wander between injections, there is a known regularity in retention time. That is, if an entity elutes in injection A at time t, then that entity will elute in another injection, B, with a retention time that will lie within a window t+Δt. That is the retention-time of a given entity can wander from one injection to another. Such wander, however, is bounded by a window t+Δt. This bound Δt can be determined empirically, and is termed herein the coarse retention time threshold, Δtc. As used herein, the term t+Δtc refers to the coarse retention time window. Although it may be the case that all entities lying within a coarse retention window have sufficiently unique masses that tracking can be done on the basis of the coarse retention time window and mass alone, in general, and especially in the case of more complex samples, there are likely entities whose mass values do not render them unique within a given coarse retention time window.